At least one in every hundred children will have a seizure. Despite this high frequency, the pathogenesis of epilepsy is incompletely understood. One malignant form of epilepsy that accounts for approximately 15% of pediatric epilepsy is known as Infantile Spasm (IS). While a number of disorders and mutations in specific genes are associated with IS, mutations in one transcription factor, ARX, are a common cause. Poly-A tracts are encoded frequently in the genome;expanded tracts in eight proteins are known to result in developmental pathology. Although it is not known how expansion of the poly-A tract in ARX leads to IS in humans, our lab has shown that expression of mouse Arx with an expanded first poly-A tract (ArxE) causes intranuclear inclusions and increased cell death in cell culture and in whole mouse brains. I hypothesize that ArxE results in dysregulation of expression as well as sequestration of the protein. This, in turn, would lead to dysregulation of expression of Arx targets, with consequences for neurodevelopment. In vitro studies including luciferase assays and chromatin immunoprecipitation will determine the functions retained and lost by ArxE and whether a subset of Arx targets are dysregulated by ArxE, and these targets will indicate pathways important for brain development. In addition, the function of the mutant protein will be investigated in in vivo experiments where ArxE is electroporated into embryonic male Arx conditional knock- out (CKO) mouse brains, and the characteristics of the CKO interneurons expressing the construct will be compared to interneurons not expressing the construct to determine functions retained, lost and gained by ArxE. Cell migration, presence of inclusions, and cell death will be analyzed. Arx transcriptional target expression levels will be measured by q-PCR to determine which pathways are altered by poly-A tract expansion on a cell-autonomous basis. Identification of the pathological mechanisms of the expanded poly- A repeat in Arx will shed light on neurodevelopment and epilepsy in general, as well as the mechanisms of diseases caused by poly-A expansions in other genes. PUBLIC HEALTH RELEVANCE: A malignant form of the common neurological disorder epilepsy is seen in patients with mutations in the gene ARX. Studying this gene's function and the problems that result from its mutation should result in insight that may improve clinical epilepsy programs.